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The rupture of the Córrego do Feijão dam in Brumadinho (January 25, 2019) caused serious damage to the Paraopeba River and compromised the quality of its waters for human consumption. However, the possible effects of the dam collapse on the river microbiome and its antibiotic resistance profiles are unknown. The present study aims to analyse the possible shifts in microbial diversity and enhancement of antibiotic resistance in the Paraopeba River. To this end, two sampling campaigns (February and May 2019) were performed to obtain water across the entire Paraopeba River (eight sampling locations: Moeda, Brumadinho, Igarapé, Juatuba, Varginha, Angueretá, Retiro Baixo and Três Marias; ~464 km). This sampling scheme enabled determining the effects of the disaster on the river microbiome. Total DNA and microbial isolation were performed with these water samples. The 16S rRNA-based microbiome analyses (n = 24; 2.05 million 16S rRNA reads) showed changes in microbial diversity immediately after the disaster with the presence of metal-indicating bacteria (Acinetobacter, Bacillus, Novosphingobium, and Sediminibacterium). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) identification of bacterial isolates (n = 170) also disclosed possible indicators of faecal contamination across the Paraopeba (Cloacibacterium, Bacteroides, Feaecalibacterium, Bifidobacterium, Citrobacter, Enterobacter, Enterococcus and Escherichia). Antibiotic resistance increased significantly to amikacin, ampicillin, ampicillin/sulbactam, amoxicillin/clavulanate, ceftriaxone, cefalotin, and meropenem among isolates obtained in May after the disaster. The effects of toxic mud on microbiomes were felt at all points sampled up to Anguereta. The ore mud may have exacerbated the growth of different antibiotic-resistant, metal-resistant, and faecal-indicating bacteria in the Paraopeba River.